bth treatment delays the senescence of postharvest pitaya
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foods
Article
BTH Treatment Delays the Senescence of Postharvest PitayaFruit in Relation to Enhancing Antioxidant System andPhenylpropanoid Pathway
Xiaochun Ding 1, Xiaoyang Zhu 2 , Wang Zheng 2, Fengjun Li 1,3, Shuangling Xiao 2 and Xuewu Duan 1,*
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Citation: Ding, X.; Zhu, X.; Zheng,
W.; Li, F.; Xiao, S.; Duan, X. BTH
Treatment Delays the Senescence of
Postharvest Pitaya Fruit in Relation to
Enhancing Antioxidant System and
Phenylpropanoid Pathway. Foods
2021, 10, 846. https://doi.org/
10.3390/foods10040846
Academic Editor: Joaquina Pinheiro
Received: 9 March 2021
Accepted: 8 April 2021
Published: 13 April 2021
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1 South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China;[email protected] (X.D.); [email protected] (F.L.)
2 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources/GuangdongProvincial Key Laboratory for Postharvest Science and Technology of Fruit and Vegetables, College ofHorticulture, South China Agricultural University, Guangzhou 510642, China;[email protected] (X.Z.); [email protected] (W.Z.); [email protected] (S.X.)
3 College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing 100049, China* Correspondence: [email protected]; Tel.: +86-20-3725-2960
Abstract: The plant resistance elicitor Benzo (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester(BTH) can enhance disease resistance of harvested fruit. Nonetheless, it is still unknown whether BTHplays a role in regulating fruit senescence. In this study, exogenous BTH treatment efficiently delayedthe senescence of postharvest pitaya fruit with lower lipid peroxidation level. Furthermore, BTH-treated fruit exhibited lower hydrogen peroxide (H2O2) content, higher contents of reduced ascorbicacid (AsA) and reduced glutathione (GSH) levels and higher ratios of reduced to oxidized glutathione(GSH/GSSG) and ascorbic acid (AsA/DHA), as well as higher activities of ROS scavenging enzymes,including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD)and glutathione reductase (GR) in comparison with control fruit. Moreover, BTH treatment enhancedthe activities of phenylpropanoid pathway-related enzymes, including cinnamate-4-hydroxylase(C4H), phenylalanine ammonia-lyase (PAL) and 4-coumarate/coenzyme A ligase (4CL) and thelevels of phenolics, flavonoids and lignin. In addition, BTH treatment upregulated the expression ofHuSOD1/3/4, HuCAT2, HuAPX1/2 and HuPOD1/2/4 genes. These results suggested that applicationof BTH delayed the senescence of harvested pitaya fruit in relation to enhanced antioxidant systemand phenylpropanoid pathway.
Keywords: pitaya; BTH; ROS metabolism; phenylpropanoid pathway
1. Introduction
Pitaya (Hylocereus undatus) is a tropical and subtropical non-climactic fruit, rich invitamins, amino acids, total phenols and betaine. Pitaya is popular among consumers for itsattractive color and high-quality taste. However, the fruit is prone to water loss, wilting, rotand rapid senescence, resulting in a short storage life. Till now, techniques are developedto maintain the quality of harvested pitaya, including chemical treatments (BABA, methyljasmonate, nitric oxide) [1,2], heat shock [3] and low temperature and bagging [4].
Benzo (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) is a structural analogof salicylic acid. BTH exerts no direct bacteriostatic effect on pathogens, but induces plantsto acquire systemic immune resistance [5,6]. It has been shown that BTH enhances plant dis-ease resistance and improves the quality of horticultural products such as muskmelon [7,8],strawberry [9], apple [10] and mango [11]. BTH also delays fruit ripening in climactericmuskmelon and banana [12,13]. However, it is still unknown whether BTH regulate thesenescence of non-climacteric fruit.
ROS are generated with aerobic respiration, which are implicated in modulating di-verse important physiological process as signaling molecules. However, when subjected
Foods 2021, 10, 846. https://doi.org/10.3390/foods10040846 https://www.mdpi.com/journal/foods
Foods 2021, 10, 846 2 of 11
to environmental stress or during senescence, excessive ROS might accumulate, result-ing in oxidative damage of macromolecules, which, in turn accelerate stresses or senes-cence [14,15]. ROS level is regulated by enzymatic antioxidant system and low molecularmass antioxidants [16–20]. Most fruits have vigorous respiration after harvest, accom-panied by ROS accumulation. It has been shown that ROS accumulation and oxidativedamage to proteins play roles in fruit senescence [21–23]. Previous studies have demon-strated that the postharvest inducer enhances the antioxidant capacity of muskmelon [8],pear [24], blueberry [25] and kiwi fruit [20]. Therefore, maintenance of redox status iscrucial for delaying fruit senescence.
The phenylpropanoid pathway plays key roles in the synthesis of phenolic compounds,flavonoids, lignins and other secondary metabolites [26,27]. In the pathway, 4-coumarate-CoA ligase (4CL) and phenylalanine ammonia-lyase (PAL) account for the critical enzymes.Of the synthesized secondary metabolites, some are antioxidants and antibiotic substances,which are important for maintenance of fruit quality and resistance to fungal infection [28].It has been shown that some exogenous elicitors treatments enhance resistance againstfungi and maintains fruit quality by activation of the phenylopropanoid pathway [8,29].
The present work focused on evaluating the role of BTH in regulating the senescencein harvested pitaya fruit and further reveal the underlying mechanism in term of ROSmetabolism and phenylpropaoid pathway.
2. Materials and Methods2.1. Plant Material and Treatments
Pitaya (H. undatus cv. Bai shui jing) fruit were collected at 30 d after anthesis. Pre-liminary experiment showed that, at a concentration range of 10–100 mg L−1, applying50 mg L−1 BTH effectively postponed the harvested pitaya fruit senescence. Therefore,in this study, 50 mg L−1 BTH was used. Pitaya fruit were immersed in distilled water(containing 0.05% Tween 80) for 15 min as the control group. In the BTH treatment group,pitaya fruit were dipped in 50 mg L−1 BTH solutions (containing 0.05% Tween 80) for15 min. Each group was carried out independently in triplicate. After treatments, the fruitwere placed into 0.02 mm thick unsealed polyethylene bag and stored at room temperature(25 ± 1 ◦C). Ten fruit are packed in one polyethylene bag, and eight bags are included ineach group (BTH and control treatment).
During storage, three fruit in each group were sampled every 2 days, followed byimmediate liquid nitrogen freezing and preservation under −80 ◦C for further analysis.Each treatment was replicated three times.
2.2. Weight Loss (WL) Rate
The weights of the pitaya fruit were recorded at 2 d intervals for evaluating weightloss. The WL rate was calculated by the following formula: %WL = (original fruit weight− final fruit weight/original fruit weight) × 100%. Six fruits were used for each replicateof each treatment.
2.3. Malondialdehyde (MDA) Content
Malondialdehyde (MDA) content in pitaya peel was determined according to theMalondialdehyde Assay Kit (Beyotime, Shanghai, China). MDA content was expressed asµmol kg−1 of fresh fruit weight.
2.4. H2O2 Content
The H2O2 content was measured according to the method of Gill et al. [30]. The H2O2content was monitored at 410 nm and displayed in the manner of mmol kg−1 of freshfruit weight.
Foods 2021, 10, 846 3 of 11
2.5. Reduced Ascorbate (AsA) and Dehydroascorbate (DHA), Reduced Glutathione (GSH) andOxidized Glutathione (GSSG) Contents
AsA and GSH contents were determined as the method described by Wei et al. [31]. DHAand GSSG contents were determined using DHA and GSSG Detection Assay Kit (Beyotine,Shanghai, China). AsA and DHA contents were expressed as mg kg−1 fresh fruit weight.GSH and GSSG contents were expressed as mmol kg−1 on fruit fresh weight, respectively.
2.6. Lignin, Total Phenolic and Flavonoids Contents
Lignin, total phenolic and flavonoids contents were analyzed according to the ap-proach of Li et al. [2].
2.7. Enzymatic Activity Assays
CAT, APX and GR activities were measured for the reduction in substrates in thereaction systems at 240 nm, 290 nm and 340 nm, respectively, as the methods described inRen et al. [32], and displayed in the manner of U/mg protein. Typically, one enzyme activityunit was referred to the enzyme volume needed to cause 0.01 absorbance units change/min.SOD activity was determined by evaluating the inhibition of photochemical reduction ofnitro blue tetrazolium by the enzyme at 560 nm as the methods previously described inYou et al. [19], and expressed as U mg−1 protein. Notably, one enzyme activity unit referredto the enzyme volume required to induce 50% inhibition of the reduction. The activitiesof POD, PAL, 4CL and C4H were determined by evaluating the changes of substratesor reaction products in the reaction systems at 470 nm, 290 nm, 222 nm and 340 nm,respectively, as the methods described in Liu et al. [8], and expressed as U mg−1 protein.Typically, one enzyme activity unit indicated the enzyme volume required to induce 0.01absorbance change per one min. The Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA,USA) was utilized to determine protein concentrations within the enzyme extracts.
2.8. RNA Extraction and Real-Time Quantitative PCR
Total RNA was extracted from 1 g peel tissue using the RNeasy Plant Mini Kit (Tiangen,China). Thereafter, 1 µg of extracted total RNA was used for preparing cDNA with Reverse-iT 1ST Strand Synthesis Kit (Tiangen, China) according to the manufacturer’s instructions.Gene sequences were obtained from transcriptome data of pitaya from Gene ExpressionOmnibus (GEO) database GSE119976.
Real-time quantitative PCR was carried out by adding 2 µL of 10 ng/µL templatecDNA, 1 µL of each primer (10 µM), sterile water and 10 µL SYBRGreen qPCR Master Mixto total volume 20 µL using the ABI PRISM 7500 Sequence Detection System (AppliedBiosystems). PCR was conducted at the conditions shown below, 95 ◦C for 10 min; followedby 95 ◦C for 20 s and 58 ◦C for 40 s for 38 cycles, with HuACTIN being an internal control.Table S1 summarizes the specific primers used in qRT-PCR. Three independent biologicalreplicates were used.
2.9. Statistical Analysis
Data were expressed as the mean ± SE of three biological replicates. Differencesbetween control and BTH-treated fruit were determined by ANOVA, followed by Student’st test (* p < 0.05; ** p < 0.01) using SPSS 19.0. Principal component analysis (PCA) wasperformed using GraphPad Prism9 (GraphPad Software Inc., San Diego, CA, USA). Thedata were normalized by Z-score [Xstd = (Xi −
_X)/Sx] to ensure that the variables being
analyzed were all on similar measurement scales. Xstd is the standardized value, Xi is theoriginal value,
_X is the variable’s mean and Sx is the variable’s standard deviation.
3. Results3.1. Effect of BTH Treatment on Weight Loss and MDA Content of Harvested Pitaya Fruit
Bract yellowing is an important senescence characteristic of harvested pitaya. Thebract of the control fruit and 10 mg L−1 BTH-treated fruit turned to yellow after 10 d of stor-
Foods 2021, 10, 846 4 of 11
age, whereas those of 50 mg L−1 and 100 mg L−1 BTH-treated remained green (Figure 1A).In the following experiment, we investigated the effect of 50 mg L−1 BTH on the senescenceof harvested pitaya in relation to redox status and phenylpropanoid pathway.
Moisture plays a vital part in maintaining pitaya fruit quality, while WL can inducefruit wilting. WL was aggravated in control fruit as the storage time extended, which wasas high as 4.2% at 10 d after storage. BTH treatment reduced water loss of harvested pitaya.The water loss rate was 2.1% in the BTH-treated fruit after 10 d of storage (Figure 1B).
MDA is an important index of lipid peroxidation, which is related to oxidative stress orsenescence in organisms. Application of BTH significantly alleviated lipid peroxidation ofharvested pitaya. After 10 d of storage, the MAD contents were 3.6 and 6.0 and µmol kg−1
in BTH-treated and control fruit, respectively (Figure 1C).
Foods 2021, 10, x FOR PEER REVIEW 4 of 12
data were normalized by Z‐score [Xstd = (Xi X)/Sx] to ensure that the variables being ana‐lyzed were all on similar measurement scales. Xstd is the standardized value, Xi is the orig‐
inal value, X is the variable’s mean and Sx is the variable’s standard deviation.
3. Results
3.1. Effect of BTH Treatment on Weight Loss and MDA Content of Harvested Pitaya Fruit
Bract yellowing is an important senescence characteristic of harvested pitaya. The
bract of the control fruit and 10 mg L−1 BTH‐treated fruit turned to yellow after 10 d of
storage, whereas those of 50 mg L−1 and 100 mg L−1 BTH‐treated remained green
(Figure 1A). In the following experiment, we investigated the effect of 50 mg L−1 BTH on
the senescence of harvested pitaya in relation to redox status and phenylpropanoid path‐
way.
Figure 1. Senescence characteristics of pitaya fruit during storage at 25 °C. (A) Visual appearance
of pitaya fruit treated with 0 (control), 10, 50 and 100 mg −1 BTH after 10 d of storage. (B,C) Effect
of BTH treatment on weight loss rate (B) and malondialdehyde (MDA) content (C) in pitaya fruit
during storage. The concentration of BTH was 50 mg−1. The data are presented as the mean ± SE of
three replicates. Asterisks indicate the significant differences between the two groups (Student’s t
test, * p < 0.05; ** p < 0.01).
Moisture plays a vital part in maintaining pitaya fruit quality, while WL can induce
fruit wilting. WL was aggravated in control fruit as the storage time extended, which was
as high as 4.2% at 10 d after storage. BTH treatment reduced water loss of harvested
pitaya. The water loss rate was 2.1% in the BTH‐treated fruit after 10 d of storage
(Figure 1B).
MDA is an important index of lipid peroxidation, which is related to oxidative stress
or senescence in organisms. Application of BTH significantly alleviated lipid peroxidation
of harvested pitaya. After 10 d of storage, the MAD contents were 3.6 and 6.0 and μmol
kg−1 in BTH‐treated and control fruit, respectively (Figure 1C).
3.2. H2O2 Content and Activities of CAT, APX and SOD
The oxidative damage of biological macromolecules caused by reactive oxygen spe‐
cies (ROS) is considered to be a main cause of senescence. H2O2 content in control fruit
rapidly increased at 8 d after harvest. BTH treatment significantly reduced the accumula‐
tion of H2O2 content at the later stage of storage (Figure 2A).
Figure 1. Senescence characteristics of pitaya fruit during storage at 25 ◦C. (A) Visual appearanceof pitaya fruit treated with 0 (control), 10, 50 and 100 mg −1 BTH after 10 d of storage. (B,C) Effectof BTH treatment on weight loss rate (B) and malondialdehyde (MDA) content (C) in pitaya fruitduring storage. The concentration of BTH was 50 mg−1. The data are presented as the mean ± SEof three replicates. Asterisks indicate the significant differences between the two groups (Student’st test, * p < 0.05; ** p < 0.01).
3.2. H2O2 Content and Activities of CAT, APX and SOD
The oxidative damage of biological macromolecules caused by reactive oxygen species(ROS) is considered to be a main cause of senescence. H2O2 content in control fruit rapidlyincreased at 8 d after harvest. BTH treatment significantly reduced the accumulation ofH2O2 content at the later stage of storage (Figure 2A).Foods 2021, 10, x FOR PEER REVIEW 5 of 12
Figure 2. Effect of BTH treatment on H2O2 content (A), activities of CAT (B), APX (C) and SOD (D)
in pitaya fruit during storage. H2O2, hydrogen peroxide; CAT, catalase; APX, ascorbate peroxi‐
dase; SOD, superoxide dismutase. Asterisks indicate the significant differences between the two
groups (Student’s t test, * p < 0.05; ** p < 0.01).
CAT, APX and SOD are important antioxidant enzymes responsible for scavenging
H2O2. As shown in Figure 2B,C, CAT and APX activities slightly increased or was constant
in control fruit within the first 8 d of storage and then markedly decreased. BTH treatment
induced the increase in CAT and APX activities at the early stage of storage and main‐
tained higher levels of CAT and APX activities at the later stage of storage (Figure 2B,C).
SOD activity fluctuated at the later stage of storage, and decreased after 6 days of storage.
BTH treatment induced and maintained a higher level of SOD activity (Figure 2D).
3.3. Activities of GR and POD, Contents of GSH and AsA and Ratios of GSH/GSSG and AsA
and DHA
GR activity increased within the first 6 d of storage, and then markedly decreased.
BTH treatment resulted in higher GR activity throughout storage (Figure 3A). POD activ‐
ity tended to decrease in control fruit during storage. BTH treatment delayed the decrease
in POD activity (Figure 3B). GSH content was generally constant in control fruit during
storage. BTH treatment resulted in higher GSH level at the later stage of storage
(Figure 3C). AsA content tended to increase during storage. BTH treatment maintained
higher AsA level (Figure 3D).
Moreover, the ratios of reduced to oxidized glutathione (GSH/GSSG) and ascorbic
acid (AsA/DHA) were high in BTH‐treated fruit than in control fruit (Figure 3E,F).
Figure 2. Effect of BTH treatment on H2O2 content (A), activities of CAT (B), APX (C) and SOD (D)in pitaya fruit during storage. H2O2, hydrogen peroxide; CAT, catalase; APX, ascorbate peroxidase;SOD, superoxide dismutase. Asterisks indicate the significant differences between the two groups(Student’s t test, * p < 0.05; ** p < 0.01).
CAT, APX and SOD are important antioxidant enzymes responsible for scavengingH2O2. As shown in Figure 2B,C, CAT and APX activities slightly increased or was constant
Foods 2021, 10, 846 5 of 11
in control fruit within the first 8 d of storage and then markedly decreased. BTH treatmentinduced the increase in CAT and APX activities at the early stage of storage and maintainedhigher levels of CAT and APX activities at the later stage of storage (Figure 2B,C). SODactivity fluctuated at the later stage of storage, and decreased after 6 days of storage. BTHtreatment induced and maintained a higher level of SOD activity (Figure 2D).
3.3. Activities of GR and POD, Contents of GSH and AsA and Ratios of GSH/GSSG and AsAand DHA
GR activity increased within the first 6 d of storage, and then markedly decreased.BTH treatment resulted in higher GR activity throughout storage (Figure 3A). POD activitytended to decrease in control fruit during storage. BTH treatment delayed the decreasein POD activity (Figure 3B). GSH content was generally constant in control fruit duringstorage. BTH treatment resulted in higher GSH level at the later stage of storage (Figure 3C).AsA content tended to increase during storage. BTH treatment maintained higher AsAlevel (Figure 3D).
Moreover, the ratios of reduced to oxidized glutathione (GSH/GSSG) and ascorbicacid (AsA/DHA) were high in BTH-treated fruit than in control fruit (Figure 3E,F).
Foods 2021, 10, x FOR PEER REVIEW 6 of 12
Figure 3. Effect of BTH treatment on activities of GR (A) and POD (B), contents of GSH (C) and
AsA (D) and ratios of GSH/GSSG (E) and ASA/DHA (F) in pitaya fruit during storage. GR, gluta‐
thione reductase; POD, peroxidase; GSH, glutathione; AsA, ascorbic acid; GSSG, oxidized gluta‐
thione; DHA, oxidized ascorbic acid. Asterisks indicate the significant differences between the two
groups (Student’s t test, * p < 0.05; ** p < 0.01).
3.4. Activities of PAL, C4H and 4CL
PAL and C4H activities in control fruit showed a slight increase during storage. BTH
induced the increases in PAL and C4H activities within the first 4 d of storage and main‐
tained higher levels of PAL and C4H activities in the late storage period (Figure 4A,B).
4CL activity in control fruit initially increased slightly and then decreased during storage.
In contrast, BTH treatment initially induced the increase in 4CL activity, and then the ac‐
tivity was maintained at a high level (Figure 4C).
Figure 4. Effect of BTH treatment on activities of PAL (A), C4H (B) and 4CL (C) in pitaya fruit
during storage. PAL, phenylalanine ammonia‐lyase; C4H, cinnamate‐4‐hydroxylase; 4CL, 4‐
coumarate/coenzyme A ligase. Asterisks indicate the significant differences between the two
groups (Student’s t test, * p < 0.05; ** p < 0.01).
3.5. Total Phenols, Flavonoids and Lignin Contents
Total phenolic content slightly increased within the first 6 d of storage and then de‐
creased in control fruit. Application of BTH promoted the synthesis of total phenolic con‐
tent. Total phenolic content was higher in BTH‐treated fruit than in control fruit through‐
out storage (Figure 5A). Flavonoid content was constant over the first 6 d of storage, then
decreased in control fruit. BTH treatment resulted in increased accumulation of flavonoids
during the later stage of storage (Figure 5B). Lignin content was virtually constant in con‐
trol fruit throughout storage. Application of BTH increased the synthesis of lignin and
maintained a higher level of lignin in harvested pitaya throughout storage (Figure 5C).
Figure 3. Effect of BTH treatment on activities of GR (A) and POD (B), contents of GSH (C) andAsA (D) and ratios of GSH/GSSG (E) and ASA/DHA (F) in pitaya fruit during storage. GR,glutathione reductase; POD, peroxidase; GSH, glutathione; AsA, ascorbic acid; GSSG, oxidizedglutathione; DHA, oxidized ascorbic acid. Asterisks indicate the significant differences between thetwo groups (Student’s t test, * p < 0.05; ** p < 0.01).
3.4. Activities of PAL, C4H and 4CL
PAL and C4H activities in control fruit showed a slight increase during storage.BTH induced the increases in PAL and C4H activities within the first 4 d of storage andmaintained higher levels of PAL and C4H activities in the late storage period (Figure 4A,B).4CL activity in control fruit initially increased slightly and then decreased during storage.In contrast, BTH treatment initially induced the increase in 4CL activity, and then theactivity was maintained at a high level (Figure 4C).
Foods 2021, 10, 846 6 of 11
Foods 2021, 10, x FOR PEER REVIEW 6 of 12
Figure 3. Effect of BTH treatment on activities of GR (A) and POD (B), contents of GSH (C) and
AsA (D) and ratios of GSH/GSSG (E) and ASA/DHA (F) in pitaya fruit during storage. GR, gluta‐
thione reductase; POD, peroxidase; GSH, glutathione; AsA, ascorbic acid; GSSG, oxidized gluta‐
thione; DHA, oxidized ascorbic acid. Asterisks indicate the significant differences between the two
groups (Student’s t test, * p < 0.05; ** p < 0.01).
3.4. Activities of PAL, C4H and 4CL
PAL and C4H activities in control fruit showed a slight increase during storage. BTH
induced the increases in PAL and C4H activities within the first 4 d of storage and main‐
tained higher levels of PAL and C4H activities in the late storage period (Figure 4A,B).
4CL activity in control fruit initially increased slightly and then decreased during storage.
In contrast, BTH treatment initially induced the increase in 4CL activity, and then the ac‐
tivity was maintained at a high level (Figure 4C).
Figure 4. Effect of BTH treatment on activities of PAL (A), C4H (B) and 4CL (C) in pitaya fruit
during storage. PAL, phenylalanine ammonia‐lyase; C4H, cinnamate‐4‐hydroxylase; 4CL, 4‐
coumarate/coenzyme A ligase. Asterisks indicate the significant differences between the two
groups (Student’s t test, * p < 0.05; ** p < 0.01).
3.5. Total Phenols, Flavonoids and Lignin Contents
Total phenolic content slightly increased within the first 6 d of storage and then de‐
creased in control fruit. Application of BTH promoted the synthesis of total phenolic con‐
tent. Total phenolic content was higher in BTH‐treated fruit than in control fruit through‐
out storage (Figure 5A). Flavonoid content was constant over the first 6 d of storage, then
decreased in control fruit. BTH treatment resulted in increased accumulation of flavonoids
during the later stage of storage (Figure 5B). Lignin content was virtually constant in con‐
trol fruit throughout storage. Application of BTH increased the synthesis of lignin and
maintained a higher level of lignin in harvested pitaya throughout storage (Figure 5C).
Figure 4. Effect of BTH treatment on activities of PAL (A), C4H (B) and 4CL (C) in pitayafruit during storage. PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4-hydroxylase; 4CL,4-coumarate/coenzyme A ligase. Asterisks indicate the significant differences between the twogroups (Student’s t test, * p < 0.05; ** p < 0.01).
3.5. Total Phenols, Flavonoids and Lignin Contents
Total phenolic content slightly increased within the first 6 d of storage and thendecreased in control fruit. Application of BTH promoted the synthesis of total phenoliccontent. Total phenolic content was higher in BTH-treated fruit than in control fruitthroughout storage (Figure 5A). Flavonoid content was constant over the first 6 d ofstorage, then decreased in control fruit. BTH treatment resulted in increased accumulationof flavonoids during the later stage of storage (Figure 5B). Lignin content was virtuallyconstant in control fruit throughout storage. Application of BTH increased the synthesisof lignin and maintained a higher level of lignin in harvested pitaya throughout storage(Figure 5C).
Foods 2021, 10, x FOR PEER REVIEW 7 of 12
Figure 5. Effect of BTH treatment on contents of total phenolics (A), flavonoids (B) and lignin (C)
in pitaya fruit during storage. Asterisks indicate the significant differences between the two
groups (Student’s t test, * p < 0.05; ** p < 0.01).
3.6. Expression of HuSODs, HuAPXs, HuCATs and HuPODs
To illuminate the response of harvested pitaya fruit to BTH, we evaluated the expres‐
sion profiling of ROS metabolism‐associated genes, including four HuSODs, two HuCATs,
three HuAPXs and three HuPODs. BTH treatment upregulated the expression of
HuSOD1/3/4 genes, but downregulated the expression of the HuSOD2 gene. Except after
day‐4 and day‐6, the expression of HuCAT1 was not affected by BTH, but the expression
of HuCAT2 was upregulated by BTH. Of the analyzed three HuAPXs and three HuPODs,
the expression of HuAPX1/2 and HuPOD1/2/4 were upregulated by BTH in comparison
with the control.
4. Discussion
BTH is a synthetic plant elicitor that has similar function and structure with SA. There
is mounting evidence that BTH effectively induces disease resistance in harvested fruits,
such as muskmelons [7,8], strawberries [9], apples [10] and mangos [11]. BTH‐induced
disease resistance might be related to altered ROS metabolism, activated phenylpro‐
panoid pathway, induced pathogenesis‐related protein and accumulation of resistant sub‐
stances [33]. Recent studies have revealed that BTH also delays fruit ripening in climac‐
teric fruits, such as muskmelon and banana [12,13]. Nonetheless, it remains largely un‐
clear whether BTH is involved in the regulation of the senescence of non‐climacteric fruit.
Pitaya is a non‐climacteric fruit that undergoes senescence after postharvest, resulting in
decreased quality [34]. In the present study, BTH pretreatment obviously inhibited turn‐
ing yellow of bract and water loss of harvested pitaya fruit. Moreover, lipid peroxidation,
a senescence‐related index, was significantly reduced in harvested pitaya fruit by BTH.
Our results indicate that BTH effectively delays the senescence of harvested pitaya, which
is possibly related to maintenance of redox status.
ROS are inevitable by‐products of aerobic metabolism in organisms, which play im‐
portant roles as signaling molecules in regulating growth and development, metabolism
and response to environmental challenges. In plants, low concentrations of ROS induce
the synthesis of plant disease‐related proteins and participate in the cross‐linking and lig‐
nification of cell walls to resist fungal infections [35]. However, excessive ROS accumula‐
tion due to imbalance of production and elimination will result in oxidative damage to
macromolecules, thereby accelerating senescence [15]. Fruit senescence has been recently
proposed to be associated with ROS accumulation and protein oxidative injury [21,22,36].
Qin et al. [22] reported that protein oxidation was intensified with the proceeding of se‐
nescence in peach fruit, and low temperature inhibits ROS accumulation, alleviates pro‐
tein carbonylation and retards fruit senescence whereas H2O2 treatment results in the op‐
posite effect. Wu et al. [36] found that high oxygen concentration resulted in higher H2O2
accumulation and accelerated fruit senescence in longan fruit whereas exposure of low
oxygen has the opposite effect. In the present study, H2O2 rapidly accumulated in pitaya
fruit during the later storage, in consistence with fruit senescence. BTH treatment resulted
in the increased H2O2 level during the initial storage, which might serve as a signal mole‐
cule to induce antioxidant enzyme activities [37], but reduced H2O2 accumulation at the
Figure 5. Effect of BTH treatment on contents of total phenolics (A), flavonoids (B) and lignin (C) inpitaya fruit during storage. Asterisks indicate the significant differences between the two groups(Student’s t test, * p < 0.05).
3.6. Expression of HuSODs, HuAPXs, HuCATs and HuPODs
To illuminate the response of harvested pitaya fruit to BTH, we evaluated the expres-sion profiling of ROS metabolism-associated genes, including four HuSODs, two HuCATs,three HuAPXs and three HuPODs. BTH treatment upregulated the expression of Hu-SOD1/3/4 genes, but downregulated the expression of the HuSOD2 gene. Except afterday-4 and day-6, the expression of HuCAT1 was not affected by BTH, but the expression ofHuCAT2 was upregulated by BTH. Of the analyzed three HuAPXs and three HuPODs, theexpression of HuAPX1/2 and HuPOD1/2/4 were upregulated by BTH in comparison withthe control.
4. Discussion
BTH is a synthetic plant elicitor that has similar function and structure with SA.There is mounting evidence that BTH effectively induces disease resistance in harvestedfruits, such as muskmelons [7,8], strawberries [9], apples [10] and mangos [11]. BTH-induced disease resistance might be related to altered ROS metabolism, activated phenyl-propanoid pathway, induced pathogenesis-related protein and accumulation of resistantsubstances [33]. Recent studies have revealed that BTH also delays fruit ripening in cli-macteric fruits, such as muskmelon and banana [12,13]. Nonetheless, it remains largelyunclear whether BTH is involved in the regulation of the senescence of non-climacteric fruit.
Foods 2021, 10, 846 7 of 11
Pitaya is a non-climacteric fruit that undergoes senescence after postharvest, resulting indecreased quality [34]. In the present study, BTH pretreatment obviously inhibited turningyellow of bract and water loss of harvested pitaya fruit. Moreover, lipid peroxidation, asenescence-related index, was significantly reduced in harvested pitaya fruit by BTH. Ourresults indicate that BTH effectively delays the senescence of harvested pitaya, which ispossibly related to maintenance of redox status.
ROS are inevitable by-products of aerobic metabolism in organisms, which play im-portant roles as signaling molecules in regulating growth and development, metabolismand response to environmental challenges. In plants, low concentrations of ROS inducethe synthesis of plant disease-related proteins and participate in the cross-linking andlignification of cell walls to resist fungal infections [35]. However, excessive ROS accumu-lation due to imbalance of production and elimination will result in oxidative damage tomacromolecules, thereby accelerating senescence [15]. Fruit senescence has been recentlyproposed to be associated with ROS accumulation and protein oxidative injury [21,22,36].Qin et al. [22] reported that protein oxidation was intensified with the proceeding of senes-cence in peach fruit, and low temperature inhibits ROS accumulation, alleviates proteincarbonylation and retards fruit senescence whereas H2O2 treatment results in the oppositeeffect. Wu et al. [36] found that high oxygen concentration resulted in higher H2O2 accu-mulation and accelerated fruit senescence in longan fruit whereas exposure of low oxygenhas the opposite effect. In the present study, H2O2 rapidly accumulated in pitaya fruitduring the later storage, in consistence with fruit senescence. BTH treatment resulted inthe increased H2O2 level during the initial storage, which might serve as a signal moleculeto induce antioxidant enzyme activities [37], but reduced H2O2 accumulation at the laterstage of storage. It is suggested that the delayed senescence by BTH is related to decreasedaccumulation of ROS.
A complicated non-enzymatic and enzymatic antioxidant mechanism has evolved inplants to cope with oxidative stress [30,38,39]. Those major antioxidants are APX, SOD andCAT. SOD is responsible for catalyzing superoxide radical dismutation into H2O2. CATdirectly eliminates H2O2 while APX degrades H2O2 using AsA as the electron donor [40].In addition, GR catalyzes the reduction of oxidized glutathione, which facilitates APX tofunction [40]. Apart from antioxidant enzymes, glutathione, ascorbic acid, tocopherolsand phenolic compounds constitute the majority of non-enzymatic antioxidants in plants,which directly eliminate ROS, provide reducing power or maintain redox state of microen-vironment [18,41,42]. Numerous studies have shown that upregulated expression of SOD,CAT and APX or increased activities of antioxidant enzymes reduce ROS accumulationand therefore increase resistance to abiotic stress in plants [17–19]. BTH-induced diseaseresistance also might be related to enhanced antioxidant enzyme activities [9,32]. In thisstudy, application of BTH upregulated expression of HuSOD1/3/4, HuCAT2 and HuAPX1/2,HuPOD1/2/4 (Figure 6) and increased activities of SOD, CAT and APX (Figure 2B–D), inconsistence with the alleviated accumulation of H2O2 (Figure 2A). Moreover, the increase inGR activity (Figure 3A), contents of GSH and AsA and ratios of GSH/GSSG and ASA/DHA(Figure 3C–F) in BTH-treated fruit compared with control fruit indicated that the redoxstate was well maintained by BTH, which are important for APX to eliminate H2O2. Per-oxidase (POD) is a hemoprotein catalyzing the oxidation of a number of substrates, suchas phenols, amine compounds and hydrocarbon oxidation products, using hydrogen per-oxide as the electron acceptor. POD works with other antioxidant enzymes to eliminateexcess ROS [43,44]. Our results showed that BTH treatment up-regulated expression ofHuPOD1/2/4 and increased POD activity in harvested pitaya fruit (Figures 3B and 6). Thus,BTH treatment activated the gene expression and activities of antioxidant enzymes, andwell maintained the redox status, which might be responsible for the alleviated H2O2accumulation and the delayed senescence in harvested pitaya fruit (Figure 7). To furtherpredict the key redox parameters of enzyme activity, metabolite content and related geneexpression in the BTH-treated pitaya fruit, we performed a multivariate PCA analysisbetween BTH-treated and control fruit (Figure S1A,B). The results showed that PC1 and
Foods 2021, 10, 846 8 of 11
PC2 together accounted for 6.34% of the cumulative proportion of variance and 22 re-dox variables were significantly separated between the BTH-treated and control group(Figure S1A). In the loadings plot for our data, the activity of CAT, GR, the content ofH2O2, AsA and GSH, the ratio of AsA/DHA and GSH/GSSG, the mRNA transcripts levelsof HuSOD1, HuSOD2, HuSOD3, HuSOD4, HuCAT2, HuAPX1, HuPOD1, HuPOD2 andHuPOD4 were strongly correlated (values close to 1 or −1, p < 0.05) (Figure S1B).
Foods 2021, 10, x FOR PEER REVIEW 9 of 12
Figure 6. Expression levels of HuSODs (A–D), HuCATs (E–F), HuAPXs (G–I) and HuPODs (J–L) in
pitaya fruit treated with or without BTH during storage. HuACT was used as the reference gene.
Gene expression of the sample from 0 d was set as 1. Asterisks indicate the significant differences
between the two groups (Student’s t test, * p < 0.05; ** p < 0.01).
Figure 7. A proposed model of the involvement of BTH in the regulation of fruit senescence in
pitaya. Arrow up means promotion, arrow down indicates suppression.
The phenylpropanoid pathway is considered as a rich source of secondary metabo‐
lites, such as phenolic compounds, flavonoids, coumarins and lignins in plants. In phe‐
nylpropanoid pathway, PAL, 4CL and C4H serve as the critical enzymes, of which PAL
and C4H are the first and second rate‐limiting enzymes [45,46], whereas 4CL is the key
enzyme leading to the branch of phenylpropanoid pathway [26]. Phenols and flavonoids
are the end products of phenylpropanoid pathway, and their accumulation are closely
associated with plant resistance and senescence [47–51]. Lignin enhances the mechanical
strength of plant cell walls and prevents microbial invasion [29], and is involved in regu‐
lation of senescence [52–55]. In the present study, application of BTH promoted PAL, 4CL
and C4H activities (Figure 4), in consistence with the increased synthesis of phenols, fla‐
vonoids and lignin (Figure 5) in pitaya fruit. Li and colleagues also came to consistent
results in red pitaya [1], who found that beta‐aminobutyric acid treatment induces re‐
Figure 6. Expression levels of HuSODs (A–D), HuCATs (E–F), HuAPXs (G–I) and HuPODs (J–L) inpitaya fruit treated with or without BTH during storage. HuACT was used as the reference gene.Gene expression of the sample from 0 d was set as 1. Asterisks indicate the significant differencesbetween the two groups (Student’s t test, ** p < 0.01).
Foods 2021, 10, x FOR PEER REVIEW 9 of 12
Figure 6. Expression levels of HuSODs (A–D), HuCATs (E–F), HuAPXs (G–I) and HuPODs (J–L) in
pitaya fruit treated with or without BTH during storage. HuACT was used as the reference gene.
Gene expression of the sample from 0 d was set as 1. Asterisks indicate the significant differences
between the two groups (Student’s t test, * p < 0.05; ** p < 0.01).
Figure 7. A proposed model of the involvement of BTH in the regulation of fruit senescence in
pitaya. Arrow up means promotion, arrow down indicates suppression.
The phenylpropanoid pathway is considered as a rich source of secondary metabo‐
lites, such as phenolic compounds, flavonoids, coumarins and lignins in plants. In phe‐
nylpropanoid pathway, PAL, 4CL and C4H serve as the critical enzymes, of which PAL
and C4H are the first and second rate‐limiting enzymes [45,46], whereas 4CL is the key
enzyme leading to the branch of phenylpropanoid pathway [26]. Phenols and flavonoids
are the end products of phenylpropanoid pathway, and their accumulation are closely
associated with plant resistance and senescence [47–51]. Lignin enhances the mechanical
strength of plant cell walls and prevents microbial invasion [29], and is involved in regu‐
lation of senescence [52–55]. In the present study, application of BTH promoted PAL, 4CL
and C4H activities (Figure 4), in consistence with the increased synthesis of phenols, fla‐
vonoids and lignin (Figure 5) in pitaya fruit. Li and colleagues also came to consistent
results in red pitaya [1], who found that beta‐aminobutyric acid treatment induces re‐
Figure 7. A proposed model of the involvement of BTH in the regulation of fruit senescence in pitaya.Arrow up means promotion, arrow down indicates suppression.
The phenylpropanoid pathway is considered as a rich source of secondary metabolites,such as phenolic compounds, flavonoids, coumarins and lignins in plants. In phenyl-propanoid pathway, PAL, 4CL and C4H serve as the critical enzymes, of which PAL andC4H are the first and second rate-limiting enzymes [45,46], whereas 4CL is the key enzymeleading to the branch of phenylpropanoid pathway [26]. Phenols and flavonoids are theend products of phenylpropanoid pathway, and their accumulation are closely associated
Foods 2021, 10, 846 9 of 11
with plant resistance and senescence [47–51]. Lignin enhances the mechanical strengthof plant cell walls and prevents microbial invasion [29], and is involved in regulation ofsenescence [52–55]. In the present study, application of BTH promoted PAL, 4CL and C4Hactivities (Figure 4), in consistence with the increased synthesis of phenols, flavonoids andlignin (Figure 5) in pitaya fruit. Li and colleagues also came to consistent results in redpitaya [1], who found that beta-aminobutyric acid treatment induces resistance against rotcaused by Gilbertella persicaria via activating phenylpropanoid. It appears that the increasedsynthesis of phenols, flavonoids and lignin in BTH-treated pitaya fruit is important tomaintain redox state and increase resistance, thereby delaying postharvest pitaya fruitsenescence (Figure 7).
5. Conclusions
BTH treatment effectively delayed postharvest pitaya fruit senescence. The delayedsenescence was related to enhanced antioxidant system and phenylpropanoid pathway.BTH treatment upregulated expression of antioxidant enzyme genes, increased activitiesof antioxidant enzymes and regenerative capacity of glutathione and ascorbic acid. More-over, BTH treatment also induced PAL, C4H and 4CL activities, resulting in the increasedsynthesis of phenols, flavonoids and lignin. Nevertheless, the investigation of the mech-anism of BTH to delay pitaya fruit senescence is still needed to be further studied at themolecular level.
Supplementary Materials: The supplementary are available online at https://www.mdpi.com/article/10.3390/foods10040846/s1. Figure S1. PCA scores analysis of the metabolites, enzymes andtranscripts in antioxidant system in pitaya fruit after BTH treatment (A). Loadings plots for PCA ofthe metabolites, enzymes and transcripts in antioxidant system in pitaya fruit after BTH treatment(B). A total of 22 variables are considered, including the activities of SOD, CAT, APX, GR, POD, thecontent of H2O2, AsA and GSH, the ratio of AsA/DHA and GSH/GSSG, the mRNA transcriptslevels of HuSOD1, HuSOD2, HuSOD3, HuSOD4, HuCAT1, HuCAT2, HuAPX1, HuAPX2, HuAPX3,HuPOD1, HuPOD2 and HuPOD4. PC1 and PC2 account for 45.28% and 15.06% of the cumulativeproportion of variance respectively. Table S1. Primers for Real-time PCR.
Author Contributions: X.D. (Xiaochun Ding): conceptualization, data curation, formal analysis,investigation, methodology, software, writing—original draft, funding acquisition. X.Z., W.Z., F.L.,S.X.: investigation, formal analysis, methodology. X.D. (Xuewu Duan): project administration,supervision, funding acquisition, writing—review and editing. All authors have read and agreed tothe published version of the manuscript.
Funding: This research was funded by National Natural Science Foundation of China (32002103),Science and Technology Planning Project of Guangzhou (201804020041), China Postdoctoral ScienceFoundation funded project (2019M650218), and Guangdong Basic and Applied Basic ResearchFoundation (2020A1515110092).
Data Availability Statement: The data presented in this study are available on request from thecorresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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